TGF-β1甲基化在类风湿性关节炎Th17/Treg细胞分化平衡中的作用机制研究

TGF-β1是一种多效能细胞因子，参与Th17/Treg细胞可塑性分化，影响类风湿关节炎（RA）发生与发展，但具体作用不清楚。前期研究发现RA患者和健康人群PBMC中TGF-β1基因启动子的甲基化程度差异显著，与TGF-β1 mRNA 及其蛋白表达相关，且与RA病理程度相关。所以TGF-β1甲基化差异与 RA中Th17/Treg细胞分化平衡紊乱关系值得探讨。本项目拟运用MS-PCR方法比较检测甲基化差异CpG位点，然后运用CRISPR/dCAS9技术融合TET1定位修饰TGF-β1差异甲基化CpG位点，分析此位置与Th17/Treg细胞分化平衡的关系。最后运用EMSA方法分离与TGF-β1基因启动子热甲基化位点结合的相关转录调控因子，分析此因子的生物学特点,研究此因子如何通过转录调控因子参与对RA的调节。以上研究对以后利用TGF-β1作为靶点在RA诊断和治疗方面提供了重要理论依据。

TGF-β1 is a poly-functional factor, however，it has an obscure function in Rheumatoid arthritis (RA). This project wants to explore the role and molecular mechanisms of TGF-β1 methylation in the pathogenesis of RA by following experiment. we have detected and compared the methylation extent of TGF-β1 promoter in PBMC samples between patients and healthy people，and got the relationship with the TGF-β1 mRNA and protein expression level. The following experiment we will get the methylation difference CpG locus by MS-PCR，and use CRISPR/dCAS9 technology to explore how the TGF-β1 methylation difference CpG position regulates the balance of Th17/ Treg cells differentiation. Then we will analyze the impact of different concentration TGF-β1 with IL-10 or IL-6 on Th17/ Treg cells differentiation. At last，we get some transcriptional regulation factors which binding the hot methylation locus of TGF-β1 promoter by EMSA method，and get some information by SDS-PAGE and western-blotting technology. In all，we wish to clarify the relationship of TGF-β1 methylation state and RA，and find some related transcriptional regulation factors which binding and regulate the methylation extent of TGF-β1 promoter，these results can support the theoretical basis for finding the mechanism of causing and healing RA.